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| United States Patent |
5,316,915
|
|
Kraus
, et al.
|
May 31, 1994
|
Method for the determination of antibodies against lipocortins
Abstract
The invention relates to a method for the determination of antibodies
against lipocortins (annexins) in a body fluid of a species, using
proteins from the lipocortin family bound to a solid phase and using a
labeled bioaffinity binding partner which is directed against single
classes or a plurality of classes of immunoglobulins of this species.
| Inventors:
|
Kraus; Michael (Marburg, DE);
Romisch; Jurgen (Marburg, DE)
|
| Assignee:
|
Behringwerke Aktiengesellschaft (Marburg, DE)
|
| Appl. No.:
|
805648 |
| Filed:
|
December 12, 1991 |
Foreign Application Priority Data
| Current U.S. Class: |
435/7.95; 427/2.13; 436/506; 436/518 |
| Intern'l Class: |
G01N 033/543; G01N 033/564 |
| Field of Search: |
435/965,7.9,7.92,970,7.95
436/501,518,513,817,506
530/389.2,388.23
427/2
|
References Cited
U.S. Patent Documents
| 4434227 | Feb., 1984 | Unger | 435/7.
|
Other References
"Presence of Autoantibody for Phospholipase Inhibitory Protein,
Lipomodulin, in Patients with Rheumatic Diseases", for Proc. Natl. Acad.
Sci. USA, by Fusao Hirata, et al., pp. 3190-3194, May 1981.
"Autoantibodies to Recombinant Lipocortin-1 in Rheumatoid Arthritis and
Systemic Lupus Erythematosus", for Annals of the Rheumatic Diseases
(1989), vol. 48, pp. 843-850, by N. J. Goulding et al.
"Purification and Characterization of Six Annexins from Human Placenta" for
Biol. Chem. Hoppe-Seyler, vol. 371 pp. 383-388, May 1990, by Juergen
Roemisch, et al.
"Annexin Proteins PP4 and PP4-X", for Biochem. J. (1990) vol. 272 pp.
223-229, by Juergen Roemisch, et al.
"Enzyme Immunoassays in Diagnostic Medicine", for Bull World Health Organ.,
vol. 53, 1976, pp. 55-65, by A. Voller, et al.
Harlow, Ed Antibodies: A Laboratory Manual Cold Spring Harbor, N.Y., 1988
pp. 553-577, 621-622.
|
Primary Examiner: Saunders; David
Attorney, Agent or Firm: Finnegan, Henderson, Farabow, Garrett & Dunner
Claims
We claim:
1. A heterogeneous immunoassay method for determining IgX antibodies
directed against lipocortins, wherein X may be any of the known
Ig-classes, comprising the steps of:
a) fixing a mixture of two or more purified known lipocortions to a solid
support;
b) contacting a sample of a body fluid with the solid support to form an
antigen-IgX complex on the solid support, wherein the lipocortin antigen
of the antigen-IgX complex is fixed to the solid support of step a) and
the IgX of the antigen-IgX complex is from the body fluid of step b);
c) removing unbound sample;
d) treating the antigen-IgX complex with a labeled Ig-class specific
antibody or a mixture of labeled Ig-class specific antibodies; and
e) determining the amount of label bound to the solid support via
antigen-IgX-labeled antibody complex as a measure of the IgX content of
the sample.
2. The immunoassay method of claim 1, wherein the lipocortins are selected
from the group consisting of PP4, PP4-X, PAP III, p68,lipocortin I and II.
3. The immunoassay of claim 1, wherein the lipocortins are from the
lipocortin family from the same species from which the sample of a body
fluid is derived.
4. The immunoassay of claim 1, wherein in the lipocortins are bound to the
solid support using a solution which is buffered in the pH range from 5.5
to 9.5.
5. The immunoassay of claim 4, wherein CaCl.sub.2 is added during step a).
6. The immunoassay of claim 4, wherein the range of pH of the solution used
in step a) is from 5.5 to 7.5.
7. The immunoassay of claim 1, wherein the sample of body fluid is diluted
in a medium containing a gamma-globulin fraction from a rabbit, which has
been immunized against sheep erythrocytes.
8. The immunoassay of claim 1, wherein the label is an enzyme.
9. The immunoassay of claim 8, wherein the enzyme is peroxidase.
10. A method of preparing a solid-phase carrier for use in a heterogeneous
immunoassay method for determining IgX antibodies directed against
lipocortins, wherein X may be any of the known Ig-classes, comprising the
steps of:
incubating a solid support with a mixture of lipocortins in pH range from
5.5 to 9.5 in the presence of CaCl; and
washing to remove unbound lipocortins.
11. A heterogeneous immunoassay method for determining IgX antibodies
directed against lipocortins, wherein X may be any of the known
Ig-classes, comprising the steps of:
a) fixing one or more purified known lipocortins to a solid support;
b) contacting a sample of a body fluid with the solid support to form an
antigen-IgX complex on the solid support, wherein the lipocortin antigen
of the antigen-IgX complex is fixed to the solid support of step a) and
the IgX of the antigen-IgX complex is from the body fluid of step b);
c) removing unbound sample;
d) treating the antigen-IgX complex with a labeled Ig-class specific
antibody or a mixture of labeled Ig-class specific antibodies; and
e) determining the amount of label bound to the solid support via
antigen-IgX-labeled antibody complex as a measure of the IgX content of
the sample.
12. The immunoassay of claim 11, wherein the lipocortins are selected from
the group consisting of PP4, PP4-X, PAP III, p68, lipocortin I and II.
13. The immunoassay of claim 11, wherein the lipocortins are from the
lipocortin family from the same species from which the sample of a body
fluid is derived.
14. The immunoassay of claim 11, wherein the sample of body fluid is
diluted in a medium containing a gamma-globulin fraction from a rabbit,
which has been immunized against sheep erythrocytes.
15. The immunoassay of claim 11, wherein the label is an enzyme.
16. The immunoassay of claim 15, wherein the enzyme is peroxidase.
17. THe immunoassay of claim 11, wherein the range of pH of the solution
used in step a) is from 5.5 to 7.5.
18. A method of preparing a solid-phase carrier for use in a heterogeneous
immunoassay method for determining IgX antibodies directed against
lipocortins, wherein X may be any of the known Ig-classes, comprising the
step of:
incubating a solid support with a single lipocortin in pH range from 5.5 to
9.5 in the presence of CaCl.sub.2
washing to remove unbound lipocortins.
Description
The invention relates to a method for the determination of antibodies
against lipocortins (annexins) in a body fluid of a species, using
proteins from the lipocortin family bound to a solid phase and using a
labeled bioaffinity binding partner which is directed against single
classes or a plurality of classes of immunoglobulins of this species.
Inflammatory disorders with an autoimmunological basis are often
accompanied by dysregulation of the immune system, which may lead to the
production of autoantibodies which are not causally connected with the
pathogenesis of the disorder. Diagnostic use of secondary antibodies of
this type is possible for differential diagnosis. Secondary autoantibodies
may, however, on the other hand also lead to side effects which are of
importance for the therapy. Thus, the attachment of immune complexes to
cell surfaces with complement binding and subsequent complement activation
causes vasculitis in the vessel wall, carditis in the myocardium and
glomerulonephritis in the renal tubules.
Secondary autoantibodies of this type against proteins from the lipocortin
family have been described in rheumatic disorders (systemic lupus
erythematosus, rheumatoid arthritis and dermatomyositis) (Hirata, F. et
al., Proc. Natl. Acad. Sci. USA, 78, 3190-3194, 1981).
This lipocortin family currently comprises six very well characterized
proteins which are called PP4, PP4-X, PAP III, p68, lipocortin I and II
(or, in accordance with the new nomenclature, lipocortin or annexin V, IV,
III, VI, I and II) . Lipocortins regulate, inter alia, the release of
arachidonic acid and thus the supply of mediators of inflammation--the
lipocortins have an antiinflammatory effect.
Since these proteins have no leader sequence, they have been detected in
relatively high concentrations especially inside cells but only in traces
in body fluids. However, lipocortins have also been on cell surfaces, and
it has been shown that they are also able to have an extracellular
antiinflammatory effect.
However, in cases of chronic inflammation there is the risk of production
of autoantibodies against lipocortins, which then lead to corticoid
resistance. The determination of autoantibodies against lipocortins ought
therefore to be an important criterion for deciding the choice of the type
of therapy, especially on long-term use of corticoids.
A method for determining autoantibodies against lipocortin I in serum using
an ELISA has been disclosed (Goulding et al., Ann. Rheum. Dis. 48,
843-850, 1989). However, this method has great disadvantages. Thus, on the
one hand, only antibodies against one protein (lipocortin I) from the
lipocortin family are detected. On the other hand, the sensitivity is low
and the background is very high owing to non-specific binding in sera from
normal subjects, so that, in particular, autoantibodies of the IgG class
are scarcely detected. Finally, it has not been possible to show that sera
containing rheumatoid factor, which are to be expected precisely in the
indicated group of patients, are determined correctly. Hence the
suitability of this method as a clinical routine screening test is low.
Hence the object was to develop a rapid immunological method which is easy
to carry out for the identification of antibodies against these lipocortin
proteins.
It has now been found that both the non-specific binding of antibodies to
solid phases coated with lipocortins, and the effect of rheumatoid factor,
in heterogeneous immunoassays can be greatly reduced by suitable coating
proteins and methods, and choice of the sample buffer and conjugate
buffer. Reliable diagnostic information is to be expected only when the
presence of antibodies against all lipocortins is tested.
Hence the invention relates to a heterogeneous immunoassay for the
determination of antibodies against lipocortins, in which a mixture of the
known lipocortins is used as bioaffinity binding partner bound to the
solid phase.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1: Effect of pH and addition of Ca.sup.2+ during coating with PP4 on
the determination of IgG and IgM anti-PP4 antibodies in a pool of human
citrated plasma from normal donors. The samples were diluted 1:15 and 1:20
in a buffer system. The measurements are stated in milliextinctions.
FIG. 2: Effect on pH and addition of Ca.sup.2+ during coating with PP4 on
the determination of IgG and IgM anti-PP4 antibodies in a pool of human
sera from normal donors. The samples were diluted 1:5, 1:10 and 1:20 in a
buffer system. The measurements are stated in milliextinctions.
Heterogeneous enzyme immunoassays are known per se to the person skilled in
the art. They can be used to detect antigens and antibodies and can be
additive, such as, for example, a sandwich immunoassay, or competitive.
The bioaffinity binding partners used to detect the bound analytes are
labeled, for example, with a radioisotope, with a fluorescent or
chemiluminescent substance or, preferably, with an enzyme, in order to
detect the binding in a known manner.
Marker enzymes for enzyme immunoassays as such are known from the
literature, and alkaline phosphatase, .beta.-galactosidase and horseradish
peroxidase are preferably used, particularly preferably horseradish
peroxidase.
Solid phases for heterogeneous enzyme immunoassays are known per se to the
person skilled in the art, and preferably used are shaped articles such
as, for example, sheet-like test elements in which the solid phase is in
the form of a matrix, such as, for example, a fabric or membrane filter,
or net-like, tubes, wells, beads, stars or the like and microparticles
(particle size <1,000 nm) such as, for example, latex particles and
magnetically attractable particles.
Particularly preferred in this context are wells in the form of microtiter
plates, latex particles and magnetically attractable particles. Mjcrotiter
plates are very particularly preferred.
Materials for solid phases are -known to the person skilled in the art.
Preferably used as binding partners on the solid phase are proteins from
the lipocortin family which are of human origin (for example as described
by Romisch et al., Biol. Chem. Hoppe-Seyler 371, 383-388, 1990). Binding
partners which can also be used are lipocortins prepared by genetic
engineering and expressed by pro- or eukaryotic cells (see, for example,
Romisch et al., Biochem. J. 272, p. 223-229 (1990)).
When carrying out heterogeneous immunoassays, various solutions known per
se to the person skilled in the art are used, inter alia for washing and
dilution, and these contain, inter alia, buffers, detergents and neutral
proteins.
These substances are also used in the solutions which are used for the
coating, including all the washing steps pertaining thereto.
Buffer systems for use in enzyme immunoassays are known to the person
skilled in the art. The person skilled in the art is also aware that the
nature of the buffer system used in each case depends on the pH to be
obtained.
Detergents for use in solutions for heterogeneous enzyme immunoassays are
likewise known to the person skilled in the art (see, for example, Voller,
A. et al., Bull. World Health Organ. 53, 55-65 (1976)), and non-ionic and
zwitterionic detergents are preferably used, and polyoxyethylenes are
particularly preferred, and .sup..RTM. Tween 20 is very particularly
preferred.
Neutral proteins for use in enzyme immunoassays are known to the person
skilled in the art, and examples preferably used are serum albumins,
gelatin, chemically modified gelatin, such as, for example, polygeline,
and milk proteins such as, for example, lactoferrin; and human or bovine
serum albumin, polygeline and lactoferrin are particularly preferred; and
polygeline and lactoferrin are very particularly preferred.
The invention further relates to a method for coating solid phases for
heterogeneous immunoassays for determining antibodies against lipocortins,
where the coating is carried out with single proteins or mixtures of
proteins from the lipocortin family in a pH range of 5-10, preferably in
the pH range of 5-7, particularly preferably at pH 5.5.
To optimize the ratio of the signal of the pathological sample to the
normal sample it is preferable to choose a particular combination of pH
and additions of divalent cations.
Preferred in this connection is a method in which the coating of the solid
phase is carried out using a solution, which is buffered in a range of pH
5-10, of proteins from the lipocortin family in the presence of divalent
cations, specifically of Ca.sup.2+, preferably 0.1-100 mmol/l,
particularly preferably 1-10 mmol/l.
It is possible where appropriate for the proteins from the lipocortin
family to be applied singly or in groups to separate solid phases, which
are then incubated, separately or together, with the sample.
A preferred method entails the coating of the solid phase being carried
out, as already mentioned, by binding to an antibody which is bound to the
solid phase by adsorption or covalently, it being necessary that this
antibody originate from a species different from that from whose body
fluid antibody against the lipocortins is to be determined, in order to
avoid cross-reactions with the secondary antibody which is used to label
the antibodies adsorbed from the body fluid.
The properties of this secondary antibody should now be chosen so that it
reacts with the antibody in the sample but not with the antibody chosen
for the coating or with the lipocortins. If the intention is to determine
antibodies against lipocortins in human serum, an antibody against human
antibodies is suitable. It is immaterial to the invention whether the
antibody is polyclonal or monoclonal. It is important that it does not
cross-react with the lipocortins which have been used for the coating.
This condition is met most straightforwardly by using antibodies and
lipocortins from the same species. Furthermore, a non-specific reaction of
the antibody used for labeling with the antibodies used for the coating
can be reduced by choosing a suitable buffer medium which preferably,
apart from the buffer substances and additions such as detergents and
proteins, contains antibodies which particularly preferably originate from
the same species from which the antibodies used for the labeling were
obtained and which do not react with the antigen used for the coating.
In connection with this optimization, the solid phase is subsequently
coated in a preferred method with agents known to the person skilled in
the art, preferably employing bovine serum albumin.
The labeled binding partner can be an antibody directed against individual
groups of immunoglobulin classes.
A preferred method is one in which the labeled binding partner is an
antibody directed against IgG, IgM or the heavy chain of IgG and IgM.
These antibodies are preferably obtained by immunization of a species,
which is preferably not the species from which the body fluid originates,
with immunoglobulins of the species to be investigated, and are labeled by
methods known to the person skilled in the art.
The invention also relates to a method in which the body fluid to be
investigated is diluted with a buffer medium which preferably contains
reagents which suppress the binding of any rheumatoid factors contained in
the body fluid to antibodies in this body fluid.
If human body fluids are used for the investigation, it is possible in a
specific type of determination of antibodies against lipocortins to dilute
samples from the IgM fraction or the total immunoglobulin fraction, which
have high concentrations of rheumatoid factors (human anti-human IgG
antibodies) and a high concentration of antibodies from the IgG fraction
which are directed against the lipocortin(s) coated on the surface, in a
buffer medium which contains suitable antibodies onto which these
rheumatoid factors adsorb and are no longer able to react with the
antibodies of the IgG fraction which are adsorbed onto the lipocortins.
Preferably used for this purpose as addition to the buffer medium is a
globulin fraction from rabbit antisheep erythrocyte antibodies.
A preferred embodiment of the determination method according to the
invention is as follows:
The wells of a microtiter plate are coated with a buffered solution of a
mixture of lipocortins PP4, PP4-X, PAP III, lipocortin I and lipocortin II
at a pH of 5 to 7 in the presence of 0.1-100 mmol/l, preferably 1-10
mmol/l, particularly preferably 5 mmol/l, CaCl.sub.2. The subsequent
coating is carried out with bovine serum albumin.
The coated microtiter plates can be dried and stored under suitable
conditions, for example sealed in plasticcoated aluminum foil, for a
lengthy period without loss of activity. For the coating, a sample or a
control serum is pipetted into the wells and, after a defined incubation
time, aspirated out again.
To determine the antibodies immobilized on the solid phase, a
peroxidase-antibody (IgG and/or Igm) conjugate is pipetted and, after a
further defined incubation time, aspirated out. To determine the bound
conjugate by photometry, a substrate solution (for example OPD or TMB) is
pipetted in and the reaction is stopped after a defined incubation time
with sulfuric acid. The extinction is determined in a photometer. The
aspiration out can be followed in each case by one or more washing steps.
In another preferred embodiment, a superparamagnetic particle is used as
solid phase, and a chemiluminescent label, such as described in, for
example, EP 0 330 050, is used as detection system.
The embodiments indicated in the examples are particularly preferred.
The claims also form part of the disclosure.
The following examples serve merely to illustrate the invention and
restrict it in no way.
EXAMPLE 1
Coating of microtiter plates with lipocortins and the effect of various
conditions on the reaction with serum and plasma from a pool from normal
donors.
The lipocortin PP4 was dissolved at a concentration of 5 mg/l (lipocortins
prepared by the method of Romisch et al. (1990) Biol. Chem. Hoppe-Seyler
371, 383-388) in the following buffer systems: 0.01 mol/l of acetate
buffer, pH 5.5; 0.01 mol/l of HEPES
(N-(2-hydroxyethyl)pipera-zine-N'-[2-ethanesulfonic acid]), pH 7.5. The
solutions were mixed either with EDTA (ethylenediaminetetraacetic acid) or
with CaCl.sub.2 at a concentration of 0.005 mol/l. 0.125 ml was placed in
each well of microtiter plates (supplied by Nunc). Incubation overnight
was followed by washing several times with 0.05 mol/l tris/HCl buffer, pH
7.4, and the microtiter plates were dried in a dryer over silica gel at
room temperature. After drying they were sealed air- and moisture-tight in
aluminum-coated plastic bags for later use.
Human plasma and serum from a pool from normal donors was diluted in the
ratio 1:5, 1:10 and 1:20 in a tris buffer (0.05 mol/l tris, PH 7.4,
containing bovine serum albumin 1% (w/w), Tween 20 0.5% (w/w) and NaCl
0.045 mol/1). 0.1 ml of the previously diluted samples was pipetted into
the lipocortin-coated wells of the microtiter plates and incubated at room
temperature for 1 hour. This was followed by aspiration out of the
solution and washing three times with buffer (for example Enzygnost
washing buffer from Behringwerke, order No. OSNK). Then 0.1 ml of an
antibody conjugate (for example anti-human IgG-peroxidase conjugate, batch
No. 63 AP 002 A; or anti-human IgM peroxidase conjugate, batch No. 88041
A, Behringwerke, Marburg) diluted 1:51 in the above tris buffer was
pipetted into each well and incubated at room temperature for 1 hour. Once
again, three washes were carried out and 0.1 ml of substrate solution (for
example o-phenylenediamine substrate solution, Behringwerke, order No.
OSNK) was introduced into each well. After 30 min at room temperature, the
reaction was stopped with 1 ml of 0.5 normal sulfuric acid in each case,
and extinction at 490 nm compared with a reference wavelength of 630 nm
was measured in an ELISA photometer (for example supplied by Titertek).
The extinctions determined under the various coating conditions in the
determination of IgG and of IgM in serum and plasma are shown in FIG. 1
and 2 respectively. It is evident that human antibodies, both of the IgG
and of the IgM type, are detectible in serum and plasma from normal donors
on lipocortin-coated plates under all the coating conditions detailed
above. The decrease in the signal with increasing dilution of the sample
employed shows that this is attributable not to non-specific binding of
the conjugate but to the adsorption of IgG and IgM from the sample. This
signal, which is called background hereinafter, was more pronounced under
the chosen experimental conditions in the plasma than in the serum and was
higher with antibodies of the IgM type than with those of IgG type. In
this example, the background for the IgG determination and for the IgM
determination was always lower in the acid than in the neutral coating
system. It was possible further to reduce the background in the acid
medium by coating in the presence of divalent cations (for example
Ca.sup.2+).
EXAMPLE 2
Subsequent coating of the lipocortin-coated microtiter plates and effect on
the background signal
Microtiter plates were coated with a mixture of lipocortin I and II (5 and
1.25 mg/l respectively) as described in Example 1 at pH 5.5, 7.5 and 9.5
with the addition of 0.005 Mol/l CaCl.sub.2. After several washes in 0.05
mol/l tris/HCl buffer pH 7.4, 0.125 ml of 1% (w/w) solutions of the
following proteins in the buffer used for the lipocortin coating was
pipetted into each well: bovine serum albumin, ovalbumin, gelatin, fetal
calf serum, and 0.1% rabbit IgG and coating buffer without added protein.
After incubation at room temperature for 3 hours, the microtiter plates
were washed several times as described in Example 1 and dried. The
background signal was determined as described in Example 1 using in each
case 0.1 ml of a 1:10 dilution of a pool of human serum from normal
donors.
The results in Table 1 show that the background signal can be reduced very
substantially, both in the determination of antibodies of the IgG type and
of the Igm type, by the nature of the coating buffer and protein chosen
for the subsequent coating. In this example, BSA proved to be particularly
suitable for the determination of antibodies of the IgG type, and BSA and
fetal calf serum for the IgM determination.
EXAMPLE 3
Dependence of the Measured Signal on the Antigen Concentration During
Coating of the Plates
Microtiter plates were coated with PP4 and PP4-X in an acetate buffer (0.01
mol/l acetate, pH 5.5; 0.005 mol/l CaCl.sub.2) in the following
concentrations: 39, 78, 156, 313, 625, 1250, 2500 and 5000 .mu.g/l. The
subsequent coating was carried out as described in Example 2 with a 1%
bovine serum albumin solution in the same acetate buffer used for the
antigen coating. After the plates had been washed and dried, human serum
from a pool of normal donors and pathological sera were diluted in the
ratio 1:20 in a tris buffer (0.05 mol/l tris, pH 7.4, containing bovine
serum albumin 3% (w/w), Tween 20 0.5% (w/w) and NaCl 0.045 mol/1). The
pathological serum used for determining IgG antibodies was that of a
patient with psoriasis arthropathica, and that for determining IgM
antibodies was that of a patient with erythema exudativum multiforme. The
bound antibodies were determined as described in Example 1.
The results, detailed in Table 2, demonstrate that a maximal reaction can
be obtained, irrespective of the absolute size of the measured signal, by
suitable choice of the lipocortin concentration during the coating. Under
the experimental conditions chosen in this example, a saturation of the
signal, both of the control and of the pathological samples, was achieved
at a lipocortin concentration greater than 2 mg/l.
EXAMPLE 4
Effect of the Choice of the Buffer Solution Employed For the Sample
Dilution on the Signal/Background Ratio
Microtiter plates were coated with PP4 and lipocortin I/II mixture in an
acetate buffer at pH 5.5 as described in Example 1, and subsequently
coated with bovine serum albumin as described in Example 2. Human serum
from a pool of normal donors and the serum from a patient with systemic
lupus erythematosus were diluted in the ratio 1:20 in a tris buffer (0.05
mol/l tris, pH 7.4, containing bovine serum albumin 3% (w/w), Tween 20
0.5% (w/w)) and various concentrations of NaCl (0.045, 0.1, 0.2, 0.3, 0.4,
0.5, 0.6 and 0. 8 mol/1) . The bound antibodies of the IgG type were
determined as described in Example 1.
The results presented in Table 3 demonstrate that the composition of the
buffer solution employed for the sample dilution (for example addition of
NaCl) may lead to a considerable reduction in the background signal, while
there is less of an effect on the signal in a pathological sample. This
makes it possible to achieve an optimal signal/background ratio. In this
example, an optimum was reached at a concentration of about 0.3 mol/l by
addition of NaCl in the determination of antibodies of the IgG type
against PP4; the optimum was at about 0.2 mol/l for a mixture of
lipocortin I and lipocortin II. Hence, in the optimization of an assay
system for detecting antibodies against lipocortins not only is the
coating of the solid phase important but also the choice of a suitable
buffer for dilution and incubation of the serum on the solid phase plays a
crucial part.
EXAMPLE 5
Suppression of Non-Specific Adsorption of the Anti-human Antibody-POD
Conjugates to Proteins on the Solid Phase
Microtiter plates were coated as described in Example 1 with PP4 and PP4-X
in an acetate buffer at pH 5.5 and subsequently coated as described in
Example 2 with bovine serum albumin. Human serum from a pool of normal
donors and the sera from patients with systemic lupus erythematosus (for
the anti-PP4 determination) and with a melanoma (for the anti-PP4-X
determination) were diluted in the ratio 1:10 in a tris buffer (0.05 mol/l
tris, pH 7.4, containing bovine serum albumin 3% (w/w), Tween 20 0.5%
(w/w) and 0.9% NaCl). The difference from determination of the antibodies
against PP4 and PP4-X described in Example 1 was that a tris buffer (0.05
mol/l tris, pH 7.4, containing bovine serum albumin 1% (w/w), Tween 20
0.5% (w/w) and NaCl 0.045 mol/1) to which various concentrations (0, 0.01,
0.02, 0.03 and 0.04% w/w) of a monoclonal antibody (for example order No.
NTIDYO 80/63, Behringwerke Marburg) had been added was used for dilution
of the anti-hIgG-POD and anti-hIgM-POD conjugate concentrates.
The results in Table 4 demonstrate that the signal both for the pool of
normal sera and that for the pathological sera is reduced by increasing
addition of mouse IgG (for example monoclonal antibody, order No. NTIDYO
80/63, Behringwerke Marburg). The ratio between the two
(pathological/normal) remains approximately the same in the anti-PP4
determination,, while the numerical value increases for PP4-X, but this
probably results purely from the computation because of the low signal in
the normal sera in this case. Thus, this example shows that non-specific
interactions between the conjugated antibody and the proteins on the solid
phase can be suppressed by suitable choice of additives in conjugate
dilution media.
EXAMPLE 6
Suppression of False-positive IgM Determinations Caused by Rheumatoid
Factors
Microtiter plates were coated as described in Example I with PP4-X in an
acetate buffer at pH 5.5 and subsequently coated as described in Example 2
with bovine serum albumin. Human sera with low titers of anti-PP4-X
antibodies of the IgG and IgM type (IgG-/Igm-) and high titers only of the
IgG (IgG+/-Igm-) or Igm type (IgG-/IgM+) were previously diluted in the
ratio 1:5 in sample buffer as described in Example 4. In a first
experimental approach, the previously diluted sera were mixed with equal
volumes of a serum which contained rheumatoid factors and had been diluted
in various concentrations in sample buffer (for example rheumatoid factor
positive control BW 18683 C. No. 12; 125 IU/ml; Behringwerke, Marburg)
(final concentrations of the rheumatoid serum 1:160, 1:80, 1:40, 1:20,
1:10 and 1:5). The content of antibodies against PP4-X was determined in
0.1 ml samples of these mixtures as described in Example 1.
The results which are presented in Table 5 and which have been corrected
for the measurements in the serum containing rheumatoid factors reveal
that, in a human serum with a high titer of antibodies of the IgG type,
"rheumatoid factor" (=anti-human IgG IgM antibodies) bound to these IgG
antibodies is also detected in the IgM determination. When the titers of
anti-lipocortin IgG antibodies are low ("IgG-"), the IgM determination is
unaffected and independent of the titer of antilipocortin IgM antibodies
("IgM-"or "IgM+").
In a second experiment, the "IgG+/IgM-" serum which indicated falsely high
IgM titers with rheumatoid factor (see Table 5) was mixed as described
above with the serum containing rheomatoid factors. The final
concentration of the "IgG+/IgM-" serum was 1:10, and that of the RF serum
was 1:20. Dilution was carried out with the sample buffer described in
Example 4, which had been mixed with various proportions (1:160 to 1:10)
of a solution of the gammaglobulin fraction of rabbit anti-sheep
erythrocyte antibodies (for example Ambo-zeptor for Rapitex reagents, No.
62 MH, batch 08, Behringwerke, Marburg). The IgM titers were determined as
described above.
The results, which are likewise shown in Table 5, demonstrate that the
falsely high IgM determinations owing to binding of rheumatoid factor to
IgG on the solid phase can be neutralized by adding a solution of a
gammaglobulin fraction to the medium used for the sample dilution.
EXAMPLE 7
Screening of Sera Using a Solid Phase Coated With All Lipocortins
A panel of human sera from normal donors and from patients with
autoimmunological and/or inflammatory disorders was tested as described in
Example 6 in dilutions of 1:10 for antibodies of the IgG and IgM type
against lipocortins. The microtiter plates were coated as described in
Example 1 with PP4, PP4-X, PAP III, p68 and lipocortin I/II mixture, and
with a mixture of all lipocortins at a concentration of 5 mg/l (lipocortin
II: 1.25 mg/1), and subsequently coated as described in Example 2 with
bovine serum albumin.
The results of the determinations of the IgG and IgM anti-lipocortin
antibodies in sera from 114 normal blood donors are listed in Table 6. The
list shows the frequency of the measurements found in classes which are
divided logarithmically (in milliextinctions). The means and median of
each of the distributions agree well but differ for each coating antigen.
Among 123 samples from patients with autoinununological and/or inflammatory
disorders, 69 were found to have titers of antibodies against lipocortins
of the IgG or IgM type above the 90% interval of the "normal distribution"
determined in each case above, and these are called "positive"
hereinafter. It emerged that 38 patients had produced antibodies of the
IgG type and 25 patients had produced those of the IgM type, these
reacting with only one lipocortin. The specificity was distributed over
all the lipocortins (see Table 7). This shows that only comprehensive
screening with all lipocortins and against both antibody types is able to
detect disturbances of the function of the lipocortins caused by
anti-lipocortin antibodies.
43 positive samples were also tested on a microtiter plate coated with all
lipocortins. This revealed "positivity" in 13/20 with IgG and 7/15 with
IgM antibodies (see Table 7). This example thus demonstrates that a solid
phase coated with all lipocortins is suitable for a general method for
screening samples for antibodies both of the IgG and of the IgM type
against single lipocortins and against several lipocortins.
TABLE 1
______________________________________
Effect of the subsequent coating of microtiter plates
coated with lipocortin I/II mixture on the IgG and IgM
determination in a pool of human sera from normal donors
(Example 2).
Used for the subsequent coating were 1% solutions of
bovine serum albumin (BSA), ovalbumin (OA), gelatin (G),
fetal calf serum (FCS), and 0.1% rabbit IgG (RI) and
coating buffer without addition (-) of protein with pH
values of 5.5, 7.5 and 9.5. The serum was diluted 1:10 in
buffer for use. The measurements are stated in milli-
extinctions; Ab = antibody class.
pH for subsequent coating
Protein 5.5 7.5 9.5 Ab
______________________________________
IgG
-- 18 29 24
BSA 10 17 20
OA 250 126 92
G 21 22 19
FCS 38 55 45
RI 24 12 36
IgM
-- 70 86 93
BSA 64 74 78
OA 73 85 87
G 111 107 99
FCS 56 84 90
RI 301 160 131
______________________________________
TABLE 2
______________________________________
Dependence of the anti-PP4 and -PP4-X IgG and IgM deter-
mination in sera from normal blood donors (N) and from
patients with autoimmune diseases (P) on the concen-
tration of the coating antigen. The measurements are
stated in milliextinctions (Example 3).
Coating antigen
Concentration
PP4 PP4-X
[mg/1] N P N P Ab class
______________________________________
0.04 63 92 69 104 IgG
0.08 53 101 70 101
0.16 50 93 71 101
0.31 62 96 86 109
0.63 78 120 110 136
1.25 122 166 139 165
2.5 142 187 180 207
5.0 152 201 185 208
0.04 10 119 23 64 IgM
0.08 25 64 22 64
0.16 22 65 26 67
0.31 25 72 29 77
0.63 28 72 35 94
1.25 37 97 46 149
2.5 42 118 62 178
5.0 46 129 63 207
______________________________________
TABLE 3
______________________________________
Effect of NaCl in the sample dilution buffer on the
determination of antibodies of the IgG type against PP4
and lipocortin I/II mixture (L I/II) in a pool of sera
from normal donors (normal sera) and a serum from a
patient with systemic lupus erythematosus (pathoserum).
The measurements are stated in milliextinctions (Example 4).
Extinction Normal sera/
NaCl concen-
(490 nm-630 nm) pathosera
tration Normal sera Pathoserum ratio
[mol/1] PP4 L I/II PP4 L I/II
PP4 L I/II
______________________________________
0.045 194 152 627 848 3.2 2.5
0.1 106 11 549 791 5.2 3.1
0.2 62 85 406 587 6.5 3.3
0.3 40 76 270 439 6.8 3.0
0.4 37 64 180 352 4.9 2.6
0.5 36 62 132 282 3.7 2.1
0.6 38 61 95 218 2.5 1.6
0.8 33 49 79 134 2.4 1.5
______________________________________
TABLE 4
______________________________________
Effect of a monoclonal antibody in the conjugate dilution
buffer on the determination of IgG antibodies against PP4
and PP4-X in a pool of human sera from normal donors
(normal sera) and from patients with systemic lupus
erythematosus (PP4) and with a melanoma (PP4-X) (patho-
sera). The measurements are stated in milliextinctions
(Example 5).
Extinction Normal sera/
MAb concen-
(490 nm-630 nm) pathosera
tration Normal sera Pathoserum ratio
[mol/1] PP4 PP4-X PP4 PP4-X PP4 PP4-X
______________________________________
0 169 64 585 336 3.5 5.3
0.01 75 20 246 209 3.3 10.3
0.02 58 13 181 168 3.1 13.1
0.03 49 9 184 152 3.7 16.3
0.04 40 6 132 134 3.3 22.9
______________________________________
TABLE 5
______________________________________
Suppression of false-positive IgM determinations caused
by rheumatoid factor. Sera with low titers of anti-PP4-X
antibodies of the IgG and IgM type (IgG-/IgM-) and high
titers only of the IgG (IgG+/IgM-) or IgM type
(IgG-/IgM+), and sample buffer were mixed with various
amounts of a human serum containing rheumatoid factor
(125 IU/ml). The IgG and IgM determination without
additions, the measurements of the IgM determination
after addition of rheumatoid factor serum, and on
addition of various amounts of a gamma-globulin solution
from rabbits to a serum with a false positive reaction
with rheumatoid factor are listed. The measurements are
stated in milliextinctions (Example 6).
______________________________________
IgG-/IgM-
IgG+/IgM- IgG-/IgM+
______________________________________
Measurements without addition [mE]
IgG 43 721 37 IgM
37 91 228
IgM determination with the addition of RF serum
1:160 26 93 200
1:80 42 105 208
1:40 32 111 231
1:20 34 149 250
1:10 24 176 215
1:5 18 201 209
IgM determination
with the addition
IgG+/IgM- +
of gamma-globulin
RF serum
solution (1:20)
without 157
1:160 147
1:80 126
1:40 140
1:20 111
1:10 112
______________________________________
TABLE 6
______________________________________
Statistical analysis of the distribution of titers of
antibodies of the IgG and IgM type against single lipo-
cortins and against mixtures of lipocortins in a panel of
sera from normal donors (n = 114) in classes divided
logarithmically (milliextinctions) (Example 7).
PP4 PP4-X PAP III p68 LI/II PP4-LII
______________________________________
IgG type:
Number 114 114 114 114 114 114
Mean 42 76 47 80 46 58
Median 39 67 41 78 49 53
90% interval
90 117 105 193 109 112
IgM type:
Number 114 114 114 114 114 114
Mean 41 70 43 59 62 58
Median 40 69 43 61 63 61
90% interval
93 154 98 141 136 126
______________________________________
TABLE 7
______________________________________
Occurrence of antibodies of the IgG and IgM type against
lipocortins in sera from patients with autoimmunological
and/or inflammatory disorders. Samples classified as
positive were those whose antibody titers are above the
90% interval of the particular distribution of the
measurements on normal donors (see Table 6 and 7).
PP4-LII designates the results of the investigation of
some of these samples on a microtiter plate coated with
all lipocortins; L I/II = lipocortin I and lipocortin II
mixture (Example 7).
Total number of patients: 123
of whom positive (IgG and/or IgM type): 69
of whom found positive in each case:
Antigen
Anti-
body PP4 PP4-LII
type (.SIGMA. = 69)
PP4-X PAP III
p68 L I/II
(.SIGMA. = 43)
______________________________________
IgG 16%(11) 15% 10%(7) 2%(1) 13% 30%(13)
(10) (9)
IgM 4% (3) 4% 7%(5) 2%(1)
19% 16% (7)
(3) (13)
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